Pilr alpha interactions and methods of modifying same

ABSTRACT

Described herein is a novel receptor-ligand interaction and agents that may modify and/or block the interaction. Methods, uses, reagents and kits for the modulation of ligand activities related to its interaction with the novel receptor are disclosed. Also disclosed are therapeutic uses of reagents in treating inflammation-related disorders.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 13/921,557, filed on Jun. 19, 2013, which is a continuation of International Application No. PCT/US2011/066753 having an international filing date of Dec. 22, 2011, which claims benefit of priority applications U.S. Provisional Patent Application Ser. No. 61/510,453filed Jul. 21, 2011, and U.S. Provisional Patent Application Ser. No. 61/426,945filed Dec. 23, 2010,

SEQUENCE LISTING

A sequence listing comprising SEQ ID NOs: 1-63 is attached hereto. Each sequence provided in the sequence listing is incorporated herein by reference, in its entirety, for all purposes.

TECHNICAL FIELD

Described herein is a novel receptor-ligand interaction and agents that may modify and/or block the interaction. Methods, uses, reagents and kits for the modulation of ligand activities related to its interaction with the novel receptor are disclosed. Also disclosed are therapeutic uses of reagents in treating inflammation-related disorders. Specifically, this disclosure relates to PILRα, its ligands and methods of modulating their interaction. Also disclosed herein are methods of treating PILRα-mediated inflammation and/or diseases, e.g., pathogenic infection, autoimmune arthritis, etc.

BACKGROUND

Immune function is regulated by the balance of activating and inhibitory signals mediated by immune cell-surface receptors (Lanier, Nature Immunol. (2001) 2:23-27; Smith et al., Immunol. Rev. (2001) 181:115-125; Moretta et al., Annu. Rev. Immunol. (2001) 19:197-223; Diefenbach & Raulet, Curr. Opin. Immunol. (2003) 15:37-44.). Among these receptors, there are families that harbor extracellular regions that have a highly conserved amino-acid sequence but have different intracellular domains responsible for activation and inhibitory signaling. Thus, activating and inhibitory members of these families can generally recognize the same or very similar ligands with slightly different specificities, presumably resulting in fine-tuning of immune regulation. Since they are involved in both the activation and inhibition of immune function, they are generally referred to as paired receptor families Paired immunoglobulin-like (Ig-like) type 2 receptors (PILRs) are one of the paired receptor families Inhibitory receptors possess immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic domains and deliver inhibitory signals via tyrosine phosphatases, such as SHP-1. The ITIM-bearing receptor PILR-alpha recruits SHP-1 via its amino-terminal SH2 domain and is likely to have cellular inhibitory potential. The lack of a cytoplasmic tail and the presence of the transmembrane lysine residue in the second receptor, PILR-beta, suggest its potential activating function. See Fournier et al., J. Immunol. (2000) 165:1197-1209; Mousseau et al., J. Biol. Chem. (2000) 275:4467-4474.; Shiratori et al., J. Exp. Med. (2004) 199:525-533. Although the ligand for mouse PILR-alpha and PILR-beta was identified as CD99 (Shiratori et al., J. Exp. Med. (2004) 199:525-533), to date no human ligand has been identified for human PILRs.

Herpes simplex virus type 1 (HSV-1) is a member of the alphaherpesvirus subfamily and can cause recurrent mucocutaneous lesions on the mouth, face, or genitalia and potentially meningitis or encephalitis. Membrane glycoprotein B (gB) of HSV-1 is a second ligand for PILR-alpha (Satoh et al., Cell (2008)132:935-944). The interaction of HSV-1 gB with PILRα mediates viral entry and cell-cell fusion. Interestingly, expression of PILRα on cells enhances HSV-1 entry, whereas expression of PILRβ does not (Fan and Longnecker, J. Virol. (2010) 84(17):8664-8672). This suggests HSV-1 gB is not a ligand of PILRβ, and subtle amino acid differences between a and b play a role in ligand selectivity. Interestingly, binding of PILRα to HSV-1 gB also requires sialylated O-glycans (T53, T480) (Fan et al., J. Virol. (2009) 83(15):7384-7390). PILRα specifically associates with HSV-1 gB, but not with other HSV-1 glycoproteins, although some other envelope proteins are known to be O-glycosylated (Fan et al., J. Virol. (2009) 83(15):7384-7390).

Although PILR-alpha and PILR-beta are abundantly expressed on myeloid cells, very little is known about their role in host defense against extracellular bacterial infection or in immune function. Thus, elucidating the role for PILR-alpha and PILR-beta is a necessary prerequisite for the prevention/treatment of diseases/conditions associated with PILR-alpha and/or PILR-beta dysfunction. The present disclosure identifies sialidated glycans as necessary and sufficient for hPILRα binding. The present disclosure also provides a method and compositions, such as selective binding agents, to modulate the interactions of the PILR and its ligands. Specifically, described herein are novel reagents and methods based on the interaction of infective microorganisms and/or endogenous ligands for the prevention/treatment of diseases/conditions associated with PILR-alpha and/or PILR-beta activity.

BRIEF SUMMARY OF THE INVENTION

The present disclosure relates to various methods, uses, reagents, and kits based on modulation of the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

One embodiment of the invention relates to an agent that may block the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

Another embodiment of the present invention relates to a composition that may comprise (1) an agent that may block the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA, and (2) a pharmaceutically acceptable carrier.

Another embodiment of present invention further relates to a method for blocking the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA; the method may comprise the step of administering an effective amount of an agent that may block the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

Another embodiment of the present invention provides an interaction occurring at the cell surface and a method for blocking the cell surface interaction between a PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA may comprise contacting cells (a cell expressing PILR-alpha and/or a cell expressing a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA) with an effective amount of an agent that may block the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

Another embodiment of the present invention provides a method for inhibiting production of an inflammatory mediator by a cell, the method may comprise blocking the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

Another embodiment of the present invention relates to the use of an agent that may block the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA for the preparation of a medicament that may block the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

Another embodiment of the present invention relates to the use of an agent for treating an inflammatory disease/condition in a subject and/or for the preparation of a medicament for treating an inflammatory disease in a subject, wherein the agent may block the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

Another embodiment of the present invention relates to a method for identifying a compound capable of blocking the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA; the method may comprise measuring the binding of PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA in the presence versus the absence of an agent, wherein a lower binding of PILR-alpha to said ligand in the presence of the agent (in comparison with the absence of the agent) may be indicative that the agent is capable of blocking the interaction between PILR-alpha and said ligand.

Another embodiment of the present invention relates to a method for identifying a compound capable of inhibiting and/or decreasing inflammation; the method may comprise measuring the binding of PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA in the presence versus the absence of the agent, wherein a lower binding of PILR-alpha to the ligand in the presence of the agent may be indicative that the agent is capable of inhibiting or decreasing inflammation.

Another embodiment of the present invention provides a method of treating an inflammatory disease or condition in a subject; the method may comprise blocking the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA in the subject.

Another embodiment of the present invention relates to a use of an agent capable of blocking the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA for treating an inflammatory disease or condition in a subject.

Another embodiment of the present invention relates to a use of an agent capable of blocking the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA for the preparation of a medicament for treating an inflammatory disease or condition in a subject.

Another embodiment of the present invention relates to a composition for treating an inflammatory disease or condition in a subject comprising (1) an agent capable of blocking the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA and (2) a pharmaceutically acceptable carrier.

Another embodiment of the present invention relates to an agent that may stimulate the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

Another embodiment of the present invention relates to a composition that may comprise (1) an agent that may stimulate the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA and (2) a pharmaceutically acceptable carrier.

Another embodiment of the present invention relates to a method for stimulating the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA; the method may comprise the step of administering an effective amount of an agent that may stimulate the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

In an embodiment of the present invention, the interaction may occur at the cell surface and a method for stimulating the cell surface interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA may comprise contacting cells (a cell expressing PILR-alpha and/or a cell expressing a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA) with an effective amount of an agent that may stimulate the interaction between PILR-alpha and said ligand.

An additional embodiment of the present invention provides a method for inhibiting production of an inflammatory mediator by a cell, the method may comprise stimulating the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

A further embodiment of the present invention relates to the use of an agent that may stimulate the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA for the preparation of a medicament that may stimulate the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

An embodiment of the present invention relates to the use of an agent for treating an inflammatory disease in a subject and/or for the preparation of a medicament for treating an inflammatory disease in a subject wherein the agent may stimulate the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

Another embodiment of the present invention relates to a method for identifying a compound capable of stimulating the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA; the method may comprise measuring the binding of PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA in the presence versus the absence of an agent, wherein an elevated binding of PILR-alpha and said ligand in the presence of the agent (in comparison with the absence of the agent) may be indicative that the agent is capable of stimulating the interaction between PILR-alpha and said ligand.

Another further embodiment of the present invention relates to a method for identifying a compound capable of stimulating the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA; the method may comprise measuring PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA activity in the presence or absence of the agent, wherein an elevated PILR-alpha activity in the presence of the agent may be indicative that the agent is stimulating the interaction between PILR-alpha and said ligand.

Another embodiment of the present invention relates to a method for identifying a compound capable of inhibiting and/or decreasing inflammation; the method may comprise measuring the binding of PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA in the presence versus the absence of the agent, wherein an elevated binding of PILR-alpha and said ligand in the presence of the agent may be indicative that the agent is capable of inhibiting or decreasing inflammation.

Yet another embodiment of the present invention provides a method of identifying a compound capable of inhibiting or decreasing inflammation; the method may comprise measuring PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA activity in the presence versus the absence of the agent, wherein an elevated PILR-alpha activity in the presence of the agent may be indicative that the agent is capable of inhibiting or decreasing inflammation.

A further embodiment of the present invention provides a method of treating an inflammatory disease or condition in a subject; the method may comprise stimulating the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA in the subject.

Another embodiment of the present invention relates to a use of an agent capable of stimulating the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA for treating an inflammatory disease or condition in a subject.

Another embodiment of the present invention relates to a use of an agent capable of stimulating the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA for the preparation of a medicament for treating an inflammatory disease or condition in a subject.

Another embodiment of the present invention relates to a composition for treating an inflammatory disease or condition in a subject comprising an agent capable of stimulating the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA and a pharmaceutically acceptable carrier.

In some aspects of any of the foregoing embodiments the ligand may be any one of NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

One more embodiment of the present invention relates to variants of PILR-alpha. In one aspect, the variant comprises an alteration at a residue corresponding to R133 in SEQ ID NO:1. The alteration may be the addition, deletion or substitution of the residue. The variant may comprise a mutation corresponding R133A. In another aspect, the variant comprises an alteration at a residue corresponding to R126 in SEQ ID NO:3. The alteration may be the addition, deletion or substitution of the residue. The variant may comprise a mutation corresponding R126A. In a further aspect, the variant comprises an alteration that abrogates binding of PILRα to a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA. In a further aspect, the variant comprises an alteration that inhibits or decreases binding of PILRα to a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

An additional embodiment of the present invention relates to a binding motif for ligands of PILR-alpha. In one aspect, the ligands comprise a sialidated glycan domain. In a second aspect, the human PILR-alpha binds to a ligand is via a sialoadhesin homology domain.

In a different embodiment, at least one ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA interacts with PILR-beta. In an aspect, the ligand binds to PILR-beta but with lower affinity than to PILR-alpha.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the scope and spirit of the invention will become apparent to one skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1E illustrate that PILRα binds to mouse but not human CD99 and that transfer of mouse CD99 PKAPT motif to human CD99 restores PILRα binding. 1A: 293T cells were transfected with mouse and human CD99 expression vectors and the transfectants were stained with hPILRα-Fc (black line), mouse PILRα-Fc (black line) or control Ig (grey area). CD99 expressing cells were gated and their binding to PILRα-Fc was shown. 1B: Comparison of profiles of O-glycans from human and mouse CD99-Fc fusion proteins. O-glycans were released by reductive μ-elimination and permethylated then analyzed by MALDI-TOF MS. 1C: shows the glycosylation motifs in mouse CD99 and the variant human CD99 where the mouse PKAPT motif was inserted. See FIG. 5A in Shiratori et al., J. Exp. Med. (2004) 199(4):525-533 for the mouse CD99 sequence. 1D: illustrates the recovery of binding of human CD99 with the PKAPT motif inserted (i.e., the PKAPT motif from mouse CD99 was inserted into the human CD99) to both mouse and human PILRα. Both human and mouse PILRα bind mouse CD99 (second row) but fail to bind human CD99 (third row). 1E: 293T cells were transfected with mouse, human CD99 or human CD99 with mouse PKAPT motif inserted after T41 and the transfectants were stained with hPILRα-Fc (black line), mouse PILRα-Fc (black line) or control Ig (grey area).

FIGS. 2A to 2E illustrate novel PILRα ligands NPDC1 and COLEC12. 2A: COS7 cells were transfected with hNPDC1 and hCOLEC12 expression vectors, stained with hPILRα-AP, mPILRα-AP and control supernatant followed with AP substrate. 2B: 293T cells were transfected with mCD99, hNPDC1 and hCOLEC12, and the transfectants were stained with isotype control, hPILRα-Fc or mouse PILRα-Fc (black line). Binding to mock transfectants (grey area) represents background binding. Transfected ligand expressing cells were gated and their binding to PILRα-Fc was shown. 2C: 293T cells were transfected with human and mouse PILRα, and the transfectants were stained with hNPDC1-Fc or hCOLEC12-his (black line). Binding to mock transfectants (grey area) represents background binding. PILRα positive cells were gated and ligand fusion staining was shown. 2D: Radioligand assay was used to determine the equilibrium binding affinity of hPILRα-Fc to hNPDC1 transiently expressed on 293T cells. ¹²⁵I labeled hPILRα-Fc was allowed to bind to cells in the presence of increasing amounts of unlabeled hPILRα-Fc. The average equilibrium K_(D) from two replicate assays was 49 nM. 2E: SPR equilibrium binding analysis of hPILRα-Fc binding to immobilized hCOLEC12 is shown. The equilibrium K_(D) for hPILRα/hCOLEC12 was 1.1 uM.

FIG. 3 shows that human NPDC1 and COLEC12 are glycosylated with sialylated glycans. Comparison of MALDI-TOF MS spectra of permethylated O-glycans, released by reductive β-elimination, from human NPDC1-Fc and human COLEC12-his. Molecular ions of permethylated glycans (glycan alditols) were detected in positive ion mode, as sodium adducts (M+Na)⁺.

FIG. 4 shows that sialylated glycans on NPDC1 or COLEC12 are required for their binding to PILRα. The binding of selected proteins to hPILRα was determined by surface plasmon resonance. Human PILRα-Fc (25 ug/ml) was immobilized to a CM5 sensor chip. Fusion proteins with and without Sialidase A treatment were used as analytes (1 μM). The difference between the responses of negative control protein and the testing proteins was shown.

FIGS. 5A to 5G illustrate that a conserved Arginine site in PILRα is required for its binding to ligand. 5A: Amino acid sequence alignment of PILRα from various species, PILRβ and N-terminal of SIGLEC1. The positions of Ig fold residues are designated by an arrow above the sequence (↓) based on comparison with Igk/l and TCRb V set Ig domains. Conserved non-Ig PILRα residues are designated by an § above the sequence. SIGLEC1 residues involved in the sialic acid-binding are boxed (dashed lines). Conserved Siglec residues across the family are boxed (solid lines). Asterisks represent PILRα amino acids that are important for sialic acid interaction. Solid arrows beneath the sequence denote the positions corresponding to active sites of SIGLEC1 crystal structure (and PILRα homology model). The underlined segments designate beta strands in PILR. Black circles represent the mutational binding analysis that have been done on PILRα. The pairwise-percentage-residue identity between PILRα and SIGLEC1 was 23%. 5B: 293T cells were transfected with wild type human and mouse PILRα (grey line), or human PILRαR126A and mouse PILRα133A (black line) expression constructs, and the transfectants were stained with mCD99-Fc, hNPDC1-Fc or hCOLEC12-his, their binding to mock transfectants (grey area) was shown as background binding. PILRα positive cells were gated and ligand fusion staining was shown. 5C: 293T cells were transfected with mouse CD99, human NPDC1, human COLEC12, or HSV1 gB expression vectors, and the transfectants were stained with hPILRα-Fc or mPILRα-Fc (grey line), hPILRαR126A-Fc or mPILRαR133A-Fc (black line), their binding to mock transfectants (grey area) was shown as background binding. Transfected ligand expressing cells were gated and PILRα-Fc staining was shown. 5D-1 to 5D-2: 293T cells were infected with HSV1. Twenty-four hours later (FIG. 5D-1 ) glycoprotein B expression in HSV1 (black line) or mock (grey area) infected cells was shown; FIG. 5D-2, hPILRα-Fc or mPILRα-Fc (grey line), hPILRαR126A-Fc or mPILRαR133A-Fc (black line) binding to HSV1 infected cells was shown, their binding to mock transfectants (grey area) was shown as background binding. 5E: WT hPILRα-Fc and hPILRα R126-Fc were immobilized on a chip and their binding to hNPDC1, hCOLEC12, and mCD99 was compared. All three proteins showed strong binding to WT hPILRα (upper three dashed lines on the graph). Little or no binding was observed with mutant hPILRα, suggesting this conserved Arg site is necessary for binding of PILRα to its ligands (lower three lines on the graph with the Mut designation). 5F-1 to 5F-2: The binding of ligand fusion proteins to wild type and Arginine mutated human PILRα. The binding of selected proteins to hPILRα was determined by SPR. Human PILRα-Fc and PILRαR126A-Fc was immobilized to a sensor chip. Fusion proteins were used as analytes (1 μM). F. The binding of hPILRα-Fc or mPILRα-Fc (grey line), hPILRαR126A-Fc or mPILRαR133A-Fc (black line) to human PBMC, T cells and monocytes (FIG. 5F-1), mouse thymocyte, CD8+ T and B cells (FIG. 5F-2) was shown, the binding of isotype control to these cells (grey area) was shown as background binding. 5G: alignment of four human PILRα isoforms.

FIG. 6 illustrates the binding of PILRα-Fc to human NPDC1 and human COLEC12 expressing cells. HEK293T cells were transfected with human NPDC1, human COLEC12, human CD99 or vector. Transfected 293T cells were incubated with control immunoglobulins or the indicated PILRα constructs indicated on the top followed by an incubation with a second antibody and analyzed by flow cytometry.

FIG. 7 illustrates the PILRα-Fc binding to ligand transfectants.

FIGS. 8A to 8C illustrate the binding of (A) mouse and (B) human PILRα and the variant PILRα fusion proteins (i.e. R126A for human and R133A for mouse) to 3 different human ligands—COLEC12, CLEC4G and FceRII. FIG. 8C also shows the binding of mPILRβ to the same ligands. Note that the variant mPILRα does not bind and that mPILRβ binds to a lesser degree than mPILRα for CLEC4G and FceRII.

FIGS. 9A to 9C illustrate the binding of (A) mouse and (B) human PILRα and the variant PILRα fusion proteins (i.e. R126A for human and R133A for mouse) to 3 different human ligands—MAG, FceRII and LRRTM4. FIG. 9C also shows the binding of mPILRβ to the same ligands. Note that the variant mPILRα does not bind and that mPILRβ binds to a lesser degree than mPILRα for FcRII.

FIGS. 10A to 10C illustrate the binding of (A) mouse and (B) human PILRα and the variant PILRα fusion proteins (i.e. R126A for human and R133A for mouse) to 3 different human ligands—BR3, IL2Ra and PSS8. FIG. 10C also shows the binding of mPILRβ to the same ligands. Note that the variant mPILRα does not bind and that mPILRβ binds to a lesser degree than mPILRα for BR3.

FIG. 11 is an extension of FIGS. 6-10C and illustrates the binding of anti-ragweed (control), hPILRα, and the variant hPILRα (R126A) fusion proteins to 2 different human ligands—hNPDC1 and mCD99. Neo indicates cells transformed with a neomycin containing vector used as a negative control. Note that the variant hPILRα does not bind any ligands tested or to the control.

FIG. 12 is a table showing the various tissue and/or cell types that express each of the listed ligands.

DETAILED DESCRIPTION

The invention will now be described in detail by way of reference only using the following definitions and examples. All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide one of skill with a general dictionary of many of the terms used in this invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Practitioners are particularly directed to Sambrook et al., 1989, and Ausubel F M et al., 1993, for definitions and terms of the art. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary.

Numeric ranges are inclusive of the numbers defining the range.

Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

The headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

Definitions PILR Family Members

As used herein, “PILR” refers to paired immunoglobulin-like receptors (PILR) alpha and beta. They are related type I transmembrane receptors bearing a highly similar (83% identity) extracellular Ig-like variable (V)-type domain but divergent intracellular signaling domains. When only one of the members is being referenced, it will be designated as either PILRα or PILRβ.

Mouse PILR-alpha (mPILRα) as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 1, which includes a potential signal sequence. Human PILR-alpha (hPILRα) as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 3, which includes a potential signal sequence. Paired immunoglobulin-like receptor alpha, ITIM-containing inhibitory receptor of the PILR family that associates with SHP-1 (PTPN6) and SHP-2 (PTPN11), involved in signaling and inhibits activation-dependent calcium mobilization in monocytic cells. PILR-alpha may also be referred to as PILRα or PILRα herein.

Mouse PILR-beta (mPILRβ) as used herein refers to the protein having the amino acid sequence of SEQ ID NO:33 Human PILR-beta (hPILRβ) as used herein refers to the protein having the amino acid sequence of SEQ ID NO:35, which includes a potential signal sequence. Paired immunoglobulin like type 2 receptor beta, may play a role in transmembrane receptor protein tyrosine kinase signaling pathway. Increased expression of the gene encoding the protein is associated with acute lymphoblastic leukemias. PILR-beta may also be referred to as PILRb or PILRβ herein.

PILR Ligands

NPDC1 as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 5, which includes a potential signal sequence. Neural proliferation, differentiation and control, I (NPDC1) is specifically expressed in neural cells when they stop to divide and begin to differentiate. It may also regulate transcription, cell proliferation, neuron differentiation, and organ morphogenesis. Its expression is developmentally regulated and persists in the adult; it increases in the embryonic brain, in distinct, defined regions, and is correlated with growth arrest and terminal differentiation. NPDC1 has long hydrophobic stretch of amino acids (residues 13-29), a coiled-coil region (amino acids 93-120), a transmembrane domain (amino acids 191-207), an acidic domain (amino acids 277-307), and MAP-kinases consensus sites (amino acids 234-244) (Evrard and Rouget, (2005) J. Neuro. Res. 79:747-755). It may be clipped and exist in a soluble form.

COLEC12 as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 7. Collectin subfamily member 12 (COLEC12) is a type II transmembrane glycoprotein that binds bacteria through its lectin domain and may play a role in host defense.

ETBR as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 8, which includes a potential signal sequence. Endothelin receptor type B (ETBR) regulates angiogenesis, smooth muscle contraction, and cell migration; gene mutation correlates with melanoma, Waardenburg syndrome, and Hirschsprung disease, aberrantly expressed in asthma, atherosclerosis, and several cancers.

CLEC4G as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 10, which includes a potential signal sequence. C type lectin superfamily 4 member G (CLEC4G) is a homodimerizing protein that functions as a pathogen associated molecular pattern receptor, may play a role in cell-cell adhesion, antigen processing, and presentation. See also, Liu et al., (2004) J. Biol. Chem. 279(18):18748-58.

BR3 as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 12. BR3 is a 184-residue type III transmembrane protein expressed on the surface of B cells (Thompson, et al., (2001) Science 293:2108-2111; Yan, et al., (2001) Curr. Biol. 11:1547-1552). BR3 is expressed in a variety of disease tissue including multiple myeloma and non-Hodgkin Lymphoma (Novak, A J (2004) Blood 104:2247-2253; Novak, A J (2004) Blood 103:689-694). BR3 mediates NF-kappaB activation and IL10 production, elevated mRNA levels are associated with systemic lupus erythematosus and multiple sclerosis.

MAG as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 13, which includes a potential signal sequence. Myelin associated glycoprotein (MAG, also called SIGLEC4A) may play a role in cell adhesion. An increase in autoimmune antibody correlates with autism, demyelinating diseases, and polyneuropathies associated with paraproteinemias. Gene polymorphism is associated with schizophrenia.

IL-2Ra as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 15, which includes a potential signal sequence. Interleukin 2 receptor alpha, plays a role in regulation of T cell mediated immune response, expression is altered in several neoplasms, immune system and inflammatory diseases, Parkinson disease, asthma, and type I diabetes mellitus.

FceRII as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 17. Fc fragment of IgE low affinity II receptor, acts in thymocyte maturation, histamine secretion, and TNF production, regulates NO production in monocytes, upregulated in hypogammaglobulinaemia, Kawasaki disease, Graves thyrotoxicosis, and chronic uremia.

LRRTM4 as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 18, which includes a potential signal sequence. Leucine rich repeat transmembrane neuronal 4 may stimulate beta-secretase mediated processing of beta-amyloid-precursor protein, may play a role in brain development and is associated with Alzheimer disease. LRRTM4 contains nine leucine rich repeats.

DAG1 as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 20, which includes a potential signal sequence. Dystroglycan 1 or dystrophin-associated glycoprotein 1 (DAG1) is an extracellular matrix glycoprotein that acts in muscle contraction, may be involved in synaptic transmission and establishment of cell polarity, aberrant protein expression correlates with muscular dystrophies and several neoplasms.

APLP1 as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 22, which includes a potential signal sequence. Amyloid beta precursor like protein 1 (APLP1) is an alpha 2A adrenergic receptor binding protein that regulates proteolysis of amyloid precursor proteins, negatively regulates endocytosis; map position of corresponding gene correlates with Alzheimer disease.

PTPRN as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 24, which includes a potential signal sequence. Protein tyrosine phosphatase receptor-type N (PTPRN) is a putative transmembrane receptor protein tyrosine phosphatase that acts as an autoantigen in type 1 diabetes mellitus.

WDR31 as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 26, which includes a potential signal sequence. WD repeat domain 31 (WDR31) contains five WD domain G-beta repeats, has low similarity to S. pombe Cpc2p, which is required for normal mating, sporulation, and protein translation and is a putative receptor for S. pombe Pck2p during cell wall synthesis and morphogenesis.

PSS8 as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 27, which includes a potential signal sequence. Protease serine 8 or prostasin (PSS8) is a serine protease that plays a role in regulation of the amiloride-sensitive epithelial sodium channel, overexpressed in ovarian cancer cells; corresponding gene expression is downregulated in prostate cancers.

SIGLEC7 as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 29, which includes a potential signal sequence. Sialic acid binding Ig-like lectin 7 (SIGLEC7) is a disialoganglioside-binding inhibitory receptor in natural killer cells that may act in myelopoiesis. It interacts with the lipooligosaccharide (LOS) on the surface of Campylobacter jejuni.

IL15-RA as used herein refers to the protein having the amino acid sequence of SEQ ID NO: 31, which includes a potential signal sequence. Interleukin 15 receptor alpha (IL15-RA), the interleukin-15 (IL15)-binding subunit of the receptor, functions in signal transduction, cell proliferation, and natural killer cell survival, may be involved in the cellular defense response to various pathogens.

The term “antibody” herein is used in the broadest sense and refers to any immunoglobulin (Ig) molecule comprising two heavy chains and two light chains, and any fragment, mutant, variant or derivation thereof which so long as they exhibit the desired biological activity (e.g., epitope binding activity). Examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies and antibody fragments.

The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g, Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The “EU numbering system” or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The “EU index as in Kabat” refers to the residue numbering of the human IgG1 EU antibody. Unless stated otherwise herein, references to residue numbers in the variable domain of antibodies means residue numbering by the Kabat numbering system. Unless stated otherwise herein, references to residue numbers in the constant domain of antibodies means residue numbering by the EU numbering system (e.g., see U.S. Provisional Application No. 60/640,323, Figures for EU numbering).

The term “multispecific antibody” is used in the broadest sense and specifically covers an antibody that has polyepitopic specificity. Such multispecific antibodies include, but are not limited to, an antibody comprising a heavy chain variable domain (V_(H)) and a light chain variable domain (V_(L)), where the V_(H)V_(L) unit has polyepitopic specificity, antibodies having two or more V_(L) and V_(H) domains with each V_(H)V_(L) unit binding to a different epitope, antibodies having two or more single variable domains with each single variable domain binding to a different epitope, full length antibodies, antibody fragments such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies and triabodies, antibody fragments that have been linked covalently or non-covalently. “Polyepitopic specificity” refers to the ability to specifically bind to two or more different epitopes on the same or different target(s). “Monospecific” refers to the ability to bind only one epitope. According to one embodiment the multispecific antibody is an IgG antibody that binds to each epitope with an affinity of 5 μM to 0.001 pM, 3 μM to 0.001 pM, 1 μM to 0.001 pM, 0.5 μM to 0.001 pM, or 0.1 μM to 0.001 pM.

In the studies described herein, Applicants have demonstrated that PILR is a functional receptor for sialidated glycan ligands. Specifically, the ligand is selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.

Other Definitions

The terms “PILR gene” or “PILR nucleic acid molecule” or “polynucleotide” refers to a nucleic acid molecule comprising or consisting of a nucleotide sequence encoding a specific PILR polypeptide. Exemplary nucleotide sequences are set forth in FIG. 1A of Fournier et al., J. Immunol. (2000) 165:1197-1209 and NM_(—)013439 for human PILRα; multiple cDNAs have been identified for PILRβ (Wilson et al., (2006) Physiol. Genomics 27:201-18.) and annotated by NCBI, e.g., NM_(—)178238.1, NM_(—)178238.2, for human PILRβ.

The term “polypeptide allelic variant” refers to the polypeptide encoded by one of several possible naturally occurring alternate forms of a gene occupying a given locus on a chromosome of an organism or a population of organisms.

The term “polypeptide derivatives” refers to a polypeptide that has been chemically modified.

The term “polypeptide fragment” refers to a polypeptide that comprises a truncation at the amino terminus (with or without a leader sequence) and/or a truncation at the carboxy terminus of the polypeptide whose sequence is as defined herein. Polypeptide fragments may result from alternative RNA splicing or from in vivo protease activity. For transmembrane or membrane-bound forms of the polypeptides, preferred fragments include soluble forms such as those lacking a transmembrane or membrane-binding domain.

In preferred embodiments, truncations comprise about 10 amino acids, or about 20 amino acids, or about 50 amino acids, or about 75 amino acids, or about 100 amino acids, or more than about 100 amino acids. The polypeptide fragments so produced will comprise about 25 contiguous amino acids, or about 50 amino acids, or about 75 amino acids, or about 100 amino acids, or about 150 amino acids, or about 200 amino acids. Such polypeptide fragments may optionally comprise an amino terminal methionine residue. It will be appreciated that such fragments can be used, for example, to generate antibodies to the polypeptides.

The term “PILR fusion polypeptide” refers to a fusion of one or more amino acids (such as a heterologous peptide or polypeptide) at the amino or carboxy terminus of a PILR polypeptide. In an aspect the heterologous polypeptide is an Fc region of on IgG.

The term “polypeptide ortholog” refers to a polypeptide from another species that corresponds to an polypeptide as defined herein. For example, mouse and human PILRα polypeptides are considered orthologs of each other. For ease of reference, the human and mouse PILRα polypeptide sequences are aligned and shown in FIG. 1C of Fournier et al., (2000) supra.

The term “polypeptide variants” refers to, for example, PILRα polypeptides comprising amino acid sequences having one or more amino acid sequence substitutions, deletions (such as internal deletions and/or PILRα polypeptide fragments), and/or additions (such as internal additions and/or PILRα fusion polypeptides) as compared to the PILRα polypeptide as defined above. Variants may be naturally occurring (e.g., PILRα polypeptide allelic variants, PILRα polypeptide orthologs and PILRα polypeptide splice variants) or may be artificially constructed. Such PILRα polypeptide variants may be prepared from the corresponding nucleic acid molecules having a DNA sequence that varies accordingly from the DNA sequence as defined above for the PILRα gene. In preferred embodiments, the variants have from 1 to 3, or from 1 to 5, or from 1 to 10, or from 1 to 15, or from 1 to 20, or from 1 to 25, or from 1 to 50, or from 1 to 75, or from 1 to 100, or more than 100 amino acid substitutions, insertions, additions and/or deletions, wherein the substitutions may be conservative, or non-conservative, or any combination thereof.

In general, a polypeptide “variant” (i.e. a variant of any polypeptide disclosed herein) means a biologically active polypeptide having at least about 80% amino acid sequence identity with the corresponding native sequence polypeptide. Such variants include, for instance, polypeptides wherein one or more amino acid (naturally occurring amino acid and/or a non-naturally occurring amino acid) residues are added, or deleted, at the N- and/or C-terminus of the polypeptide. Ordinarily, a variant will have at least about 80% amino acid sequence identity, or at least about 90% amino acid sequence identity, or at least about 95% or more amino acid sequence identity with the native sequence polypeptide. Variants also include polypeptide fragments (e.g., subsequences, truncations, etc.), typically biologically active, of the native sequence.

“Percent (%) amino acid sequence identity” herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a selected sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087, and is publicly available through Genentech, Inc., South San Francisco, Calif. The ALIGN-2 program should be compiled for use on a UNIX operating system, e.g., digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

For purposes herein, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

-   100 times the fraction X/Y where X is the number of amino acid     residues scored as identical matches by the sequence alignment     program ALIGN-2 in that program's alignment of A and B, and where Y     is the total number of amino acid residues in B. It will be     appreciated that where the length of amino acid sequence A is not     equal to the length of amino acid sequence B, the % amino acid     sequence identity of A to B will not equal the % amino acid sequence     identity of B to A.

The term “heterologous” when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not normally found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences, e.g., from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source Similarly, a heterologous protein will often refer to two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

“Essentially pure” protein means a composition comprising at least about 90% by weight of the protein, based on total weight of the composition, preferably at least about 95% by weight, more preferably at least about 90% by weight, even more preferably at least about 95% by weight. “Essentially homogeneous” protein means a composition comprising at least about 99% by weight of protein, based on total weight of the composition.

The term “antagonist” when used herein refers to a molecule capable of neutralizing, blocking, inhibiting, abrogating, reducing or interfering with the activities of a protein of the invention including its binding to one or more receptors in the case of a ligand or binding to one or more ligands in case of a receptor. Antagonists include antibodies and antigen-binding fragments thereof, proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleic acids, bioorganic molecules, peptidomimetics, pharmacological agents and their metabolites, transcriptional and translation control sequences, and the like. Antagonists also include small molecule inhibitors of a protein of the invention, and fusions proteins, receptor molecules and derivatives which bind specifically to protein thereby sequestering its binding to its target, antagonist variants of the protein, antisense molecules directed to a protein of the invention, RNA aptamers, and ribozymes against a protein of the invention.

A “blocking” antibody or an “antagonist” antibody is one which inhibits or reduces biological activity of the antigen it binds. Certain blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

The term “antigen” refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody, and additionally capable of being used in an animal to produce antibodies capable of binding to an epitope of each antigen. An antigen may have one or more epitopes.

The terms “active” and “biologically active” polypeptides refer to polypeptides having at least one activity characteristic of the reference polypeptide. For example, the enzymatic activity associated with a protease is proteolysis and, thus, an active protease has proteolytic activity. It follows that the biological activity of a given protein refers to any biological activity typically attributed to that protein by those of skill in the art. as defined above.

As used herein, the term “immunoadhesin” designates antibody-like molecules that combine the binding specificity of a heterologous protein (an “adhesin”) with the effector functions of immunoglobulin constant domains. Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity that is other than the antigen recognition and binding site of an antibody (i.e., is “heterologous”), and an immunoglobulin constant domain sequence. The adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand—such as a PILRα, for example. The immunoglobulin constant domain sequence in the immunoadhesin can be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD, or IgM.

In one aspect thereof, the present invention relates to an agent that may block the interaction between a ligand (as defined herein) and a PILR family member. In another aspect, the present invention relates to an agent that may modulate the interaction between a ligand (as defined herein) and a PILR family member.

As used herein, an “agent” that may block the interaction between a ligand (as defined herein) and PILRα may be a protein. For example, such protein may be an (isolated) antibody, or antigen-binding fragment (portion) thereof, that may specifically bind to a ligand (as defined herein) and/or PILRα. The antibody may be, for example, a monoclonal antibody and/or a polyclonal antibody. Monoclonal antibodies (MAbs) may be made by one of several procedures available to one of skill in the art, for example, by fusing antibody producing cells with immortalized cells and thereby making a hybridoma. The general methodology for fusion of antibody producing B cells to an immortal cell line is well within the province of one skilled in the art. Another example is the generation of MAbs from mRNA extracted from bone marrow and spleen cells of immunized animals using combinatorial antibody library technology. One drawback of MAbs derived from animals or from derived cell lines is that although they may be administered to a patient for diagnostic or therapeutic purposes, they are often recognized as foreign antigens by the immune system and are unsuitable for continued use. Antibodies that are not recognized as foreign antigens by the human immune system have greater potential for both diagnosis and treatment. Methods for generating human and humanized antibodies are now well known in the art.

Polyclonal antibodies may be obtained by immunizing a selected animal with a protein or polypeptide (for example without limitation a ligand or PILRα or PILRβ). Serum from the animal may be collected and treated according to known procedures. Polyclonal antibodies to the protein or polypeptide of interest may then be purified by affinity chromatography. Techniques for producing polyclonal antisera are well known in the art.

Antibodies may originate for example, from a mouse, rat or any other mammal. The antibody may also be a human antibody which may be obtained, for example, from a transgenic non-human mammal capable of expressing human immunoglobulin genes. The antibody may also be a humanized antibody which may comprise, for example, one or more complementarity determining regions of non-human origin. It may also comprise a surface residue of a human antibody and/or framework regions of a human antibody. The antibody may also be a chimeric antibody which may comprise, for example, variable domains of a non-human antibody and constant domains of a human antibody. Suitable antibodies may also include, for example, an antigen-binding fragment, a Fab fragment; a F(ab′)2 fragment, and Fv fragment; or a single-chain antibody comprising an antigen-binding fragment (e.g., a single chain Fv). An antibody encompassed in the present invention may be an antibody binding specifically to PILRα. In an embodiment, an antibody encompassed in the present invention may be an antibody binding specifically to a ligand (as described herein).

Anti-PILR agents (e.g. antibodies) may be experimentally tested and validated using in vivo and in vitro assays. Suitable assays include, but are not limited to, activity assays and binding assays.

The activity of a ligand interaction with PILR may, among other means, be measured by the following methods:

Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3. 1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann, et al., Proc. Natl. Acad. Sci. USA 78:2488-2492 (1981); Herrmann, et al., J. Immunol. 128:1968-1974 (1982); Handa, et al., J. Immunol. 135:1564-1572 (1985); Takai, et al., I. Immunol. 137:3494-3500 (1986); Takai, et al., J. Immunol. 140:508-512 (1988); Bowman, et al., J. Virology 61:1992-1998; Bertagnolli, et al., Cellular Immunology 133:327-341 (1991); Brown, et al., J. Immunol. 153:3079-3092 (1994).

Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Th1/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol. 144:3028-3033 (1990); and Assays for B cell function: In vitro antibody production, Mond, J. J. and Brunswick, M. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.

Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Th1 and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai, et al., J. Immunol. 137:3494-3500 (1986); Takai, et al., J. Immunol. 140:508-512 (1988); Bertagnolli, et al., J. Immunol. 149:3778-3783 (1992).

Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol. 134:536-544 (1995); Inaba et al., J. Exp. Med. 173:549-559 (1991); Macatonia, et al., J. Immunol. 154:5071-5079 (1995); Porgador, et al., J. Exp. Med. 182:255-260 (1995); Nair, et al., J. Virology 67:4062-4069 (1993); Huang, et al., Science 264:961-965 (1994); Macatonia, et al., J. Exp. Med. 169:1255-1264 (1989); Bhardwaj, et al., J. Clin. Invest. 94:797-807 (1994); and Inaba, et al., J. Exp. Med. 172:631-640 (1990).

Assays for lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808 (1992); Gorczyca, et al., Leukemia 7:659-670 (1993); Gorczyca, et al., Cancer Res. 53:1945-1951 (1993); Itoh, et al., Cell 66:233-243 (1991); Zacharchuk, J. Immunol. 145:4037-4045 (1990); Zamai, et al., Cytometty 14:891-897 (1993); Gorczyca, et al., Int. J. Oncol. 1:639-648 (1992).

Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica, et al., Blood 84:111-117 (1994); Fine, etal., Cell. Immunol. 155:111-122, (1994); Galy, et al., Blood 85:2770-2778 (1995); Toki, et al., Proc. Nat. Acad. Sci. USA 88:7548-7551 (1991) .

According to the present invention, a (protein) agent may also be a “soluble protein”. Soluble proteins (purified) of the invention may be obtained from any techniques well known in the art. For example, a soluble protein may be obtained by transfecting a recombinant DNA molecule expressing solely the extracellular region of a molecule and/or portion thereof followed by purification. In another example, a protein and/or a portion of a protein (for example an extracellular region exempt of its transmembrane and cytoplasmic domains) may be fused to a constant domain (Fc portion) of an immunoglobulin. A (purified) soluble protein of the present invention may be soluble PILR and/or portion thereof. By “portion” (of soluble protein for example) it is meant a portion that exhibits similar (biological) activity yet is smaller in size. An agent of the present invention may be soluble PILRα. An agent of the present invention may be portions of soluble PILRα. Human PILRα (SEQ ID NO: 3) is a 303 amino acid protein. Its extracellular domain is approximately 170 amino acids in length. A (purified) soluble human PILRα may have a sequence that may consist from about residue 20 to residue 207 of SEQ ID NO:3. The present invention relates to and explicitly incorporates herein each and every specific member and combination of sub-ranges therein whatsoever. Thus, any specified range or group is to be understood as a shorthand way of referring to each and every member of a range or group individually as well as each and every possible sub-ranges or sub-groups encompassed therein; and similarly with respect to any sub-ranges or sub-groups therein.

As used herein, the term “block” or “inhibit” refers to a decrease in one or more given measurable activity by at least 10% relative to a reference and/or control. Where inhibition is desired, such inhibition is preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, up to and including 100%, i.e., complete inhibition or absence of the given activity. As used herein, the term “substantially inhibits/blocks” refers to a decrease in a given measurable activity by at least 50% relative to a reference. For example, “substantially inhibits” refers to a decrease in a given measurable activity of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and up to and including 100% relative to a reference. As used herein, “blocks/prevents/inhibits/impairs/lowers the interaction”, with reference to the binding of a ligand that binds to a receptor refers to a decrease in binding by at least 10% relative to a reference. An agent may block the binding of a ligand to a receptor-expressing cells “Inhibits the interaction” and/or “block the binding” preferably refers to a decrease in binding of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, up to and including 100%. A “receptor” as provided for herein means PILRα or PILRβ. A “ligand” as provided for herein is selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA. A general feature of a ligand is glycan modification, e.g., sialidated glycans.

A “composition” of the invention including an agent may be manufactured in a conventional manner. In particular, it is formulated with a pharmaceutically acceptable diluent or carrier, e.g., water or a saline solution such as phosphate buffer saline. In general, a diluent or carrier is selected on the basis of the mode and route of administration, as well as standard pharmaceutical practice. Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it may be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of compositions may be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin. Moreover, an agent of the invention may be administered in a time release formulation, for example in a composition which includes a slow release polymer. The active agents may be prepared with carriers that will protect the agent against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG). Many methods for the preparation of such formulations are patented or generally known to those skilled in the relevant art. The present invention relates to compositions that may comprise an agent capable of modulating PILR (e.g., PILRα or PILRβ) activity and a pharmacologically acceptable carrier. In one embodiment, such compositions include an agent that may block the interaction between a ligand and PILRα to treat an PILRα-related disease (for example an immune-related disease and/or inflammatory disease and/or microbial infection).

As used herein “pharmaceutically acceptable carrier” or excipient includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In one embodiment, the carrier is suitable for parenteral administration. Alternatively, the carrier may be suitable for intravenous, intraperitoneal, intramuscular, sublingual or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media is incompatible with the active agent, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds may also be incorporated into the compositions.

“Administration” of a composition may be performed by any suitable routes. Such routes may include parenteral, pulmonary, nasal and/or oral routes. In one embodiment, the pharmaceutical composition may be intra-muscular (IM), subcutaneous (SC), intra-dermal (ID), intra-venous (IV) and/or intra-peritoneal (IP) routes using any suitable means.

The term “effective amount” is intended to mean an amount of an agent sufficient to substantially block the interaction between a ligand (as defined herein) and PILR (e.g., PILRα or PILRβ). An effective amount may also encompass either “therapeutically effective amount” and/or “prophylactically effective amount”. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as a reduction in disease progression and/or alleviation of the symptoms associated with a disease. A therapeutically effective amount of modulators of a PILR activity may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the agent to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the agent are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as preventing and/or inhibiting (reducing) the rate of disease onset or progression. A prophylactically effective amount may be determined as described above for the therapeutically effective amount. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering of the compositions.

During inflammation, various molecules may be secreted by cells. Such molecules may be referred to as “inflammatory mediators”. As will be appreciated by one skilled in the art, these inflammatory mediators may be, for example and without limitation, amines, eicosanoids, growth factors, reactive oxygen species, enzymes (for example a proteinase), chemokines, cytokines, etc.

Measuring the binding of a ligand (as defined herein) to PILRα may be performed using (without limitation) such suitable assays as quantitative comparisons comparing kinetic and equilibrium binding constants. The kinetic association rate (k_(on)) and dissociation rate (k_(off)), and the equilibrium binding constants (K_(d)) may be determined using surface plasmon resonance on a BIAcore™ instrument following the standard procedure in the literature. Binding properties of these interactions may also be assessed by flow cytometry and/or by solid phase binding assay.

The present invention also relates to a method of identifying a compound capable of blocking the interaction between a ligand and PILRα; the method may comprise measuring a ligand-mediated PILRα activity in the presence or absence of the agent, wherein a lower PILRα activity in the presence of the agent may be indicative that the agent is blocking the interaction between a ligand and PILRα.

As used herein, “an activity mediated by a ligand” or “a ligand-mediated PILRαactivity” is an activity involving or resulting from the binding of a ligand to PILRα, and includes, but is not limited to, binding to PILRα, the induction of T cells to produce and secrete cytokines (for example IL-2, IL-10, IFN-γ and TNF-α), the synthesis of inflammatory molecules (inflammatory mediators) such as IL-6, IL-8 and metalloproteinases and T-cell proliferation (or inhibition thereof), etc. It will be understood that the ligand-mediated activity may depend on the specific ligand, e.g., NPCD1, BR3, etc., being evaluated.

In an embodiment the use may be for the treatment or prevention of inflammatory-related diseases or condition, or a microbial infection in the subject.

One of skill in the art will recognize that while the some definitions have been written with reference to a specific polypeptide they are equally applicable to other polypeptides described herein.

Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in the relevant art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Furthermore, numeric ranges are inclusive of the numbers defining the range. In the claims, the word “comprising” is used as an open-ended term, substantially equivalent to the phrase “including, but not limited to”. The following examples are illustrative of various aspects of the invention, and do not limit the broad aspects of the invention as disclosed herein.

In the experimental disclosure which follows, the following abbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N (Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); g (grams); mg (milligrams); kg (kilograms); μg (micrograms); L (liters); ml (milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ° C. (degrees Centigrade); h (hours); min (minutes); sec (seconds); msec (milliseconds); Ci (Curies) mCi (milliCuries); μCi (microCuries); TLC (thin layer achromatography); CIA (Collagen Induced Arthritis); EAE (Experimental allergy encephalitis); AIA (Antibody induced arthritis); DTH (Delayed Type Hypersensitivity)

EXAMPLES

The present invention is described in further detain in the following examples which are not in any way intended to limit the scope of the invention as claimed. The attached Figures are meant to be considered as integral parts of the specification and description of the invention. All references cited are herein specifically incorporated by reference for all that is described therein. The following examples are offered to illustrate, but not to limit the claimed invention.

Cells and Reagents used in the Examples were as follows: All transfections were performed with Fugene 6 (Roche) or Lipofectamine 2000 (Invitrogen) according to the manufacturers protocol. Mouse total RNA adult tissue panel was purchased from Zyagen (San Diego, Calif.). Recombinant human cytokines were from Peprotech (Rocky Hill, N.J.). The following anti-mouse antibodies were used for flow cytometry: anti-mPILRα, anti-mCD99 and anti-mCOLEC12 are all from R&D Systems (Minneapolis, Minn.), anti-FLAG (M2) monoclonal antibody was purchased from Sigma-Aldrich and labeled with Alexa Flour-647 monoclonal antibody labeling kit from Invitrogen, and anti-mouse IgG2a-FITC was from BD Biosciences (San Jose, Calif).

Example 1 Recombinant Production of PILR, Ligands and Antibodies Thereof

This example illustrates preparation of potentially glycosylated forms of the desired ligands or PILR proteins (either of which is referred to in this example as a desired protein) by recombinant expression in mammalian cells. Preparations of PILR antibodies are also described.

The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), was employed as the expression vector in all instances. Optionally, DNA encoding the desired protein was ligated into pRK5 with selected restriction enzymes to allow insertion of such DNA using ligation methods such as described in Sambrook et al., supra. Epitope-tagged variants of the desired protein may also be expressed in cells. The DNA encoding the desired protein was ligated into pRK5 containing the desired epitope tag (poly-His, FLAG, human IgG₁ Fc) in frame with the desired epitope tag.

The predicted extracellular domains of human and mouse PILR were cloned without the transmembrane domain into the pRK5 vector containing a C-terminal human IgG₁-Fc or 8×-His tag, or with the transmembrane domain into the pRK5 vector containing a C-terminal GFP tag.

Soluble forms of these proteins were produced in a CHO cell transient transfection and purified by affinity chromatography using anti-FLAG (M2) agarose affinity gel (Sigma-Aldrich) for FLAG-tagged proteins, Ni-NTA agarose (Qiagen) for 8×-His-tagged proteins, or protein-A Sepharose (Amersham Pharmacia) for IgG₁-Fc fusion proteins. Proteins were further separated from aggregates and contaminants with a Superdex 200 gel-filtration column and/or MonoQ/S ion exchange columns (Amersham). Protein purity was assessed by SDS-PAGE followed by SimplyBlue Safe Stain (Invitrogen) and purified proteins were aliqouted and frozen at −80° C. until needed.

In one embodiment, the selected host cells may be HEK293T cells. Human 293 cells (ATCC CCL 1573) were grown to 50-80% confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics. 1-10 μg of DNA encoding the desired protein ligated into pRK5 was introduced into HEK293T cells using commercially available transfection reagents Superfect® (Qiagen), Lipofectamine® (Invitrogen) or Fugene® (Roche) according to manufacturer's instructions. 18-24 hours after the transfections, the culture medium was removed and tested in selected bioassays or cells were harvested using 10 mM EDTA in 20 mM Na phosphate buffer, pH7.4, and tested in selected bioassays.

Stable expression of the desired protein was achieved in HEK293T cells by cloning DNA encoding the desired protein into pRK5 vector with a selection marker that confers resistance to the antibiotic Geneticin®. For stable expression of desired proteins, cells were transfected as described and allowed to grow in DMEM with a concentration of Geneticin® that would permit growth of cells in which the desired vector had integrated into the genome (1-0.5 μg/ml).

In another embodiment, the epitope tagged versions of the desired protein can be expressed in host CHO cells. Twelve micrograms of the desired plasmid DNA was introduced into approximately 10 million CHO cells using commercially available transfection reagents Superfect® (Qiagen), Dosper®, Lipofectamine® (Invitrogen) or Fugene® (Boehringer Mannheim) according to manufacturer's instructions. The cells are grown as described in Lucas et al. (Nucl. Acids Res. (1996) 24:9 1774-1779). Approximately 3×10⁻⁷ cells are frozen in an ampule for further growth and production as described below.

The ampules containing the plasmid DNA are thawed by placement into a water bath and mixed by vortexing. The contents are pipetted into a centrifuge tube containing 10 mLs of media and centrifuged at 1000 rpm for 5 minutes. The supernatant was aspirated and the cells were resuspended in 10 mL of selective media (0.2 μm filtered PS20 with 5% 0.2 μm diafiltered fetal bovine serum). The cells are then aliquoted into a 100 mL spinner containing 90 mL of selective media. After 1-2 days, the cells are transferred into a 250 mL spinner filled with 150 mL selective growth medium and incubated at 37° C. After another 2-3 days, 250 mL, 500 mL and 2000 mL spinners are seeded with 3×10⁵ cells/mL. The cell media was exchanged with fresh media by centrifugation and resuspension in production medium. Although any suitable CHO media may be employed, a production medium described in U.S. Pat. No. 5,122,469, issued Jun. 16, 1992 may actually be used. A 3 L production spinner was seeded at 1.2×10⁶ cells/mL. On day 0, the cell number and pH was determined On day 1, the spinner was sampled and sparging with filtered air was commenced. On day 2, the spinner was sampled, the temperature shifted to 33° C., and 30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (e.g., 35% polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion) taken. Throughout the production, the pH was adjusted as necessary to keep it at around 7.2. After 10 days, or until the viability dropped below 70%, the cell culture was harvested by centrifugation and filtering through a 0.22 μm filter. The filtrate was either stored at 4° C. or immediately loaded onto columns for purification.

For the poly-His tagged constructs, the proteins are purified using a Ni-NTA column (Qiagen). Before purification, imidazole was added to the conditioned media to a concentration of 5 mM. The conditioned media was pumped onto a 6 ml Ni-NTA column equilibrated at 4° C., in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. After loading, the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The purified protein was then run over a Superdex S200 gel filtration column and/or a MonoQ/S ion exchange column (Applied Biosystems) to remove aggregated or proteolysed protein or any contaminants and subsequently concentrated and dialyzed into PBS. The homogeneity was assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation. Proteins were stored at −80° C. until used in bioassays.

For the FLAG-epitope tagged constructs, the proteins are purified using an anti-FLAG (M2) agarose column (Sigma). The conditioned media was pumped onto a 6 ml anti-FLAG column equilibrated at 4° C. with 20 mM Na phosphate buffer, pH 7.4. After loading, the column was washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein was immediately neutralized by collecting 1 ml fractions into tubes containing 275 μL of 1 M Tris buffer, pH 9. The highly purified protein was subsequently run over size exclusion chromatography, dialyzed, analyzed, and stored as above for the poly-His tagged proteins.

Immunoadhesin (Fc-containing) constructs are purified from the conditioned media as follows. The conditioned medium was pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 7.4. After loading, the column was washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein was immediately neutralized by collecting 1 ml fractions into tubes containing 275 μL of 1 M Tris buffer, pH 9. The highly purified protein was subsequently run over size exclusion chromatography, dialyzed, analyzed, and stored as above for the poly-His tagged proteins.

4 to 6-week-old Armenian hamsters (Cytogen) or mice were immunized with 2 μg/injection each murine and human recombinant PILRα proteins. The immunogens are resuspended in monophosphoryl lipid A/trehalose dicorynomycolate adjuvant and injected via footpad or I.P. at 3 to 4 day intervals for a total of 10 boosts. Three days after the final boost, lymphocytes from immunized hamster spleens and lymph nodes were harvested for fusion with SP2/0 myeloma cells (American Type Culture Collection) by using the Cyto Pulse CEEF-50 apparatus (Cyto Pulse Sciences). Briefly, after washing twice with Cytofusion Medium C (Cyto Pulse Sciences), the isolated lymphocytes and SP2/0 cells were mixed at a 1:1 ratio and then resuspended at 10 million cells/ml in Cytofusion Medium C, electrofusion was performed according to manufacturer's guidance. Fused cells were cultured in ClonaCell-HY Medium C (StemCell Technologies) overnight at 37° C. in a 7% CO₂ incubator. The next day, fused cells were centrifuged and resuspended in 10 ml ClonaCell-HY Medium C and then gently mixed with 90 ml Methylcellulose-based ClonaCell-HY Medium D (StemCell Technologies) containing HAT components. The fused cells were plated into 100 mm Petri dishes (Becton Dickinson) and allowed to grow in 37° C. in a 7% CO₂ incubator. After 7-10 days incubation, the single hybridoma clones were picked by ClonePix (Genetix, United Kingdom) and transferred into 96-well cell culture plates (Becton Dickinson) with 200 μL/well ClonaCell-HY Medium E (StemCell Technologies). Hybridoma culture media was changed prior to ELISA screening. All ELISA positive clones were further screened by FACS. After at least 2 rounds of single cell subcloning by limiting dilution, final clones were scaled up and the supernatants were collected for antibody purification. The hybridoma supernatants were purified by Protein A affinity chromatography, then sterile filtered (0.2 μm pore size, Nalgene Nunc International, NY, USA) and stored at 4° C. in PBS. The purified mAbs were confirmed by ELISA and FACS before testing on functional assays. The isotypes of purified mAbs were determined by the mouse monoclonal antibody isotyping kit from Roche Diagnostics Corporation. The isotypes of purified hamster mAbs were determined by ELISA.

Example 2 Sialidation of PILRα Ligands

This example uses one PILRα ligand, mCD99, to illustrate the importance of sialidation on the ligand for PILRα binding. Mouse PILRα binds to mCD99 with relatively low affinity, however, it is unclear whether PILRα can bind human CD99. Shiratori et al. (2004) J Exp Med 199, 525-533; Tabata et al. (2008) J Biol Chem 283, 8893-8901. To test this, we expressed mouse or human CD99 in 293T cells to see whether they can bind to PILRα-mIgG2a (PILRα-Fc) fusion proteins. We found both mouse and human PILRα-Fc fusions bound mCD99 transfectants. However, neither protein bound to hCD99 transfectants (FIG. 1A). This suggests that the CD99 interaction with PILRα is not conserved at least across human and mouse species. However a conserved PILRα interaction domain between mouse and human PILRα could mediate its binding to mCD99.

The differential binding of mouse and human CD99 to PILRα may be the result of the low sequence identity between CD99 homologs (about 41% in the extracellular domain) or the differences in glycosylation (type and/or a number of glycans). It has been shown that sialylated O-linked glycans in mCD99 play an essential role in PILRα binding to mCD99. Wang et al. (2008) J Immunol 180, 1686-1693. hCD99 has been reported to be O-glycosylated, and similarly to mCD99, does not have any potential N-glycosylation sites.

Gelin et al. (1989) EMBO J 8, 3253-3259. Lack of binding of PILRα to hCD99 may therefore be related to the differences in its O-glycosylation pattern. To examine this possibility, we first compared profiles of O-glycans from human and mouse CD99-Fc fusion proteins. O-glycans were released by reductive β-elimination and permethylated prior to analysis by MALDI-TOF MS. Comparison of MALDI-TOF spectra (FIG. 1B), demonstrated qualitatively similar O-glycosylation profiles of human and mouse CD99. Two major mono- and di-sialylated O-glycans, having the following compositions: NeuAc₁Hex₁HexNAc₁ and NeuAc₂Hex₁HexNAc₁, were detected in both proteins. The composition of observed O-glycans is consistent with the presence of sialylated Galβ1-3GalNAc core 1 structures. North et al. (2010) J Biol Chem 285, 5759-5775; Olson et al. (2005) Glycobiology 15, 177-191.

Since O-glycans of human and mouse CD99 did not differ qualitatively, we next examined whether there might be a difference in the number of O-glycans present on each of the proteins. LC-MS analysis of reduced fusion proteins (with and without PNGase F and Sialidase digestion) showed that hCD99-Fc carried two O-glycans and mCD99-Fc carried three O-glycans with the following composition: NeuAc₁₋₂Hex₁HexNAc₁. In the case of mCD99, two adjacent O-glycosylation sites, Thr-45 and Thr-50 (NMKPT ⁴⁵PKAPT ⁵⁰PKKPS) are relevant for PILRα recognition. Wang et al. (2008). Sequence alignment of human and mouse CD99 showed that Thr-41 of hCD99 corresponds to the Thr-45 0-glycosylation site of mCD99 and that hCD99 lacks the second potential O-glycosylation site corresponding to Thr-50 of mCD99. Therefore, we performed LC-MS tryptic peptide mapping of hCD99-Fc to determine the localization of O-glycans and to examine whether the peptide containing Thr-41 is indeed O-glycosylated. Two sialylated glycopeptides with following sequence were detected: APDGGFLDLSDALPDNENKKPTAIPK (hCD99-derived) and GPTIKPCPPCK (mIgG2a Fc-derived). Each glycopeptide was glycosylated with a single NeuAc₁₋₂Hex₁HexNAc₁ O-glycan. The presence of two sialylated glycopeptides was consistent with the LC-MS analysis of reduced hCD99-Fc. The detected sialoglycopeptide APDGGFLDLSDALPDNENKKPT ⁴¹AIPK contained Thr-41 corresponding to Thr-45 O-glycosylation site of mCD99 and a Ser residue which potentially could be O-glycosylated. However, the exact site of O-glycosylation could not have been sequenced through the performed type of LC-MS experiment. The presence of an O-glycan on the GPTIKPCPPCK peptide is consistent with the previous report of it being O-glycosylated in the truncated version of mouse IgG2a. Masuda et al. (1999) Mol Immunol 36, 993-1003.

Our results indicated that human and mouse CD99 have similar O-glycans but that the presence of a common core of 1 NeuAc₁₋₂Hex₁HexNAc₁ O-glycan per se on hCD99 is not sufficient for PILRα binding. A major difference between the mouse and human CD99 O-glycan modifications is that mCD99 has two O-glycosylation sites (Thr-45 and Thr-50) versus one in the human counterpart. Wang et al. (2008). We asked whether the introduction of the mCD99 region P⁴⁶KAPT⁵° (PKAPT) into hCD99 can confer the binding of PILRα to hCD99. We expressed full-length mCD99, hCD99 and hCD99PKAPT in 293T cells, and again tested the binding of hPILRα-Fc or mPILRα-Fc by FACS. We found that the insertion of mCD99 PKAPT motif into hCD99, directly following the Thr-41 O-glycosylation site, was able to induce binding of human or mouse PILRα to hCD99PKAPT expressing cells. Surprisingly, this interaction was similar in magnitude to human or mouse PILRα binding to mCD99. This suggests the sialylated O-glycans are required for CD99 binding to PILRα (FIG. 1C-E).

Example 3 Identification of Novel PILRα Ligands

This example demonstrates the identification of novel ligands for PILRα.

It has been shown that mCD99, HSV1 glycoprotein B and PANP are PILRα ligands. CD99 is expressed on T-cells, B-cells, NK cells, monocytes and neutrophils. However, hPILRα is expressed predominately in cells of the myelomonocytic lineage, including monocytes/macrophages, granulocytes, and dendritic cells (DC). Discordant PILRα-Fc binding and CD99 expression as well as a low affinity interaction with CD99 (2.2 mM) suggested that there may be additional ligands for PILRα. To identify novel PILRα ligands and to further investigate the nature of PILRα interactions with its ligands as well as biological functions thereof, a cDNA expression library was screened with an Alkaline phosphatase (AP)-tagged PILRα construct.

The extracellular domains of hPILRα (Met1-196Thr) and mPILRα (Met1-197Val) were cloned into the expression vector pRK5 as fusions to C-terminal alkaline phosphatase (AP) tag. 2931 cells were transfected with PILRα-AP-pRK5 constructs and Fugene 6 Transfection Reagent (Roche) according to the manufacturer's instructions. Three days after transfection, supernatants were collected for screening. COS7 cells were plated in a 24-well format and transfected with 10 ul of Origene library DNA (Origene, human DNA library containing 20 k genes) per well with Fugene 6 Reagent. Two days later, cells were incubated with human or mouse PILRα-Ap supernatants for 45 min at room temperature (RT). Then cells were fixed with 4% PFA for 15 min at RT. After removing fixation buffer, cells were blocked with 100 mM Glycine in HBS (20 mM Hepes, PH7.2, 150 mM NaCl) for 15 min at RT. Cells were then rinsed and incubated in HBS for 90 min at 65° C. HBS was removed and Western Blue Substrate (Promega) was added and color was developed for 30 min to 1 hr.

The resulting positive clones were identified and further analyzed using FACS. The ligands that bound hPILRα are NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA. See FIGS. 6-11. The expression of these newly identified PILRα ligands on various cell types are summarized in FIG. 12.

The hPILRα-AP binding to human NPDC1 and human COLEC12 expressing cells are subject to further analysis (FIG. 2A) Similar to mCD99, both mouse or human PILRα-AP were able to bind to hNPDC1 and hCOLEC12 expressing cells suggesting that a conserved interaction domain mediates PILRα binding to these ligands. NPDC1 is a type I transmembrane protein and has been identified as a neural-specific gene involved in the control of cell proliferation and differentiation. Galiana et al. (1995) Proc Natl Acad Sci USA 92, 1560-1564. COLEC12 is a type II transmembrane collectin family member as also known as collectin placenta 1 (CL-P1) and Scavenger Receptor with C-type Lectin (SRCL) type I. Ohtani et al. (2001) J Biol Chem 276, 44222-44228; Nakamura et al. (2001) Biochem Biophy Res Commun 280, 1028-1035.

To confirm the binding of PILRα to hNPDC1 and hCOLEC12 on the cell surface, N-terminal flag-tagged hNPDC1 or C-terminal his tagged hCOLEC12 were expressed in 293T cells, and then tested for mPILRα-Fc and hPILRα-Fc binding. Mock transfected cells were used as a negative control and mCD99 transfected cells were used as positive control. Both mPILRα-Fc and hPILRα-Fc bound to mCD99, hNPDC1 and hCOLEC12 expressing cells (FIG. 2B). Similar results were also obtained by hNPDC1-Fc or hCOLEC12-his in PILRαtransfectants by FACS analysis. Again, both hNPDC1-Fc and hCOLEC12-his bound to hPILRα as well as mPILRα tranfectants, (FIG. 2C). Using an equilibrium competition radioligand assay, the affinity (K_(D)) of hPILRα-Fc binding to cell-surface expressed hNPCD1 was determined to be 49 nM (FIG. 2D). Because the sensitivity of the radioligand competition assay was too low to measure the lower affinity interaction between hPILRα-Fc and hCOLEC12, we used SPR instead (FIG. 2E). Using this method, the equilibrium K_(D) of this interaction was found to be 1.1 uM. Taken together these studies demonstrate specific binding of PILRα to human NPDC1 and COLEC12.

The presence of sialylated O-glycans is required for PILRα binding to its known ligands including mCD99, HSV1-gB and the recently identified PANP. Kogure et al. (2011) Biochem Biophys Res Commun 405, 428-533; Wang et al. (2008); Wang et al. (2009) J Virol 83, 13042-13045. Human NPDC1 and COLEC12 have multiple potential O-glycosylation sites when analyzed using the NetOGlyc 3.1 prediction server. Julenius et al. (2005) Glycobiology 15, 153-164. hNPDC1 does not have any potential N-glycosylation sites, while hCOLEC12 has multiple sites. To determine whether both proteins are indeed O-glycosylated, they were analyzed in similar manner to mouse and human CD99. MALDI-TOF MS analysis of permethylated O-glycans released by reductive β-elimination from hNPDC1-Fc and hCOLEC12-his confirmed the presence of O-glycans, qualitatively similar to those determined in mouse and human CD99 (FIG. 3). In addition, analysis of hCOLEC12 by a combination of HPLC charge profiling of fluorescent 2-aminobenzoic acid labeled glycans and MALDI-TOF of permethylated unlabeled glycans also demonstrated its complex N-glycosylation profile. Neutral glycans were mostly of the high-mannose type (predominantly Man-5) and complex afucosylated or core-fucosylated bi-, tri- and tetra-antennary glycans, galactosylated to various degrees. Negatively charged glycans were sialylated (0-4 NANA), complex type bi-, tri- and tetra-antennary N-glycans, core-fucosylated and mostly fully galactosylated.

In order to test whether sialylated glycans on NPDC1 and COLEC12 are required for their binding to PILRα, we performed Surface Plasmon Resonance analysis using Biacore with hNPDC1 and hCOLEC12 fusion proteins with and without Sialidase A treatment. Before Sialidase A treatment, the proteins showed good binding affinity to hPILRα (FIG. 4). However, after Sialidase A treatment the proteins showed little or no binding to hPILRα (FIG. 4). mCD99 was used as a control and showed similar results to both hNPDC1 and hCOLEC12 (FIG. 4). These studies suggest that the sialyated glycans on hNPDC1 and hCOLEC12 are required for their binding to hPILRα.

Our binding analysis indicates that hNPDC 1 binds with higher affinity (K_(D)=49 nM) than hCOLEC12 (K_(D)=1 μM). Although PILRα binds to NPDC1 with relative higher affinity, both hPILRα and mPILRα do not bind to mNPDC1 Similar to CD99, differences in NPDC1 glycosylation may be responsible for this observation. NPDC1 has been identified as a neuronal-specific gene involved in the control of cell proliferation and differentiation. Galiana et al. (1995) Proc Natl Acad Sci USA 92, 1560-1564. Our results suggest that PILRα recognizes several ligands, some of which are not conserved between mouse and human. The identification of multiple PILRα ligands in the neuronal system (PANP and NPDC1) suggests that PILRα might play a role in the CNS.

COLEC12 is the only member of the Collectin scavenger receptor family that is expressed as a cell surface transmembrane protein and its ECD contains coiled-coil, collagen-like, and C-type lectin/carbohydrate domains. Ohtani et al. (2001) J Biol Chem 276, 44222-44228. COLEC12 is expressed in vascular endothelia cells and monocytes to mediate the uptake of oxidized low density lipoprotein and microbes. Ohtani et al. (2001) J Biol Chem 276, 44222-44228; Nakamura et al. (2001) Biochem Biophys Res Commun 280, 1028-1035. Interestingly, Collectins have been shown to interact with other inhibitory receptors such as SIRPa to modulate lung pathophysiology. Janssen et al. (2008) Am J Respir Crit Care Med 178, 158-167. PILRα is emerging as a receptor that recognizes a specific group of ligands in cellular or pathogenic sources with unique sialic acid pattern. Correspondingly, we found that human and mouse PILRα-Fc fusions bind to mouse thymocytes, peripheral CD8+ T and activated CD4+ T, B, NK, NKT cells, granulocytes and monocytes, as well as a majority of human PBMC. These data suggest that PILRα ligands are broadly expressed in immune cells. Since the presence of sialylated glycans is a common feature of all known PILRα ligands, it is apparent that PILRα has additional cellular ligands.

Example 4 Conserved PILRα Domain for its Interaction with Diverse Sialvlated Ligands

This example demonstrates a conserved domain on PILRα critical for its binding to sialylated ligands.

Sialylated glycan modifications appear to be a general feature of all identified PILRα ligands and coincidently few other receptors such as Siglecs. Kogure et al., (2011) Biochem Biophys Res Commun 405, 428-433; Wang et al. (2008); Crocker et al. (2007) Nat Rev Immunol 7, 255-266; Wang et al. (2009). A conserved Arg site in the ECD of Siglecs plays a critical role in their binding to sialic acid. May et al. (1998) Mol Cell 1, 719-728; Ikehara et al. (2004) J Biol Chem 279, 43117-43125; Vinson et al. (1996) J Biol Chem 271, 9267-9272. The PILRα ECD has two, similar Arg sites (human Arg 96, mouse arginine 103; human R126, mouse R133) that are highly conserved across species (FIG. 5A). The second Arg site (mouse R133, human R126) corresponds to the critical Arg site that is required for the binding of Siglecs to sialic acid in the ligands. May et al. (1998) Mol Cell 1, 719-728. The hPILRα R126 is not an Ig fold stabilizing residue (FIG. 5A). We examined whether mutation of this Arg site in mouse or human PILRα affects their binding to ligands. First, we tested the binding of ligand fusion proteins including mCD99-Fc, hNPDC1-Fc and hCOLEC12-his to cell surface-expressed WT or Arg mutated hPILRαR126A and mPILRαR131A. The Arg mutation did not affect cell surface expression of human and mouse PILRα. We found that all fusion proteins bound to wild type human and mouse transfectants, but none of them bound to hPILRαR126A and mPILRαR133A transfectants (FIG. 5B), suggesting Arg126 in hPILRα and Arg133 in mouse PILRα are required for PILRα binding to ligands. We also generated WT and Arg mutant human and mouse PILRα-Fc fusion proteins, and examined their binding to cell surface expressed ligands. The result showed that WT human and mouse PILRα-Fc bound to mCD99, hNPDC1, hCOLEC12 and HSV-1 gB transfectants, while hPILRαR126A-Fc and mPILRαR133A-Fc did not bind to these ligand transfectants (FIG. 5C). Glycoprotein B expression could be detected on HSV1 infected cells 24 hrs after infection (FIG. 5 D-1), we therefore tested wild type and Arg mutant human and mouse PILRα-Fc binding to HSV1 infected cells. Again, we observed the binding of only WT hPILRα-Fc and mPILRα-Fc but not hPILRαR126A and mPILRαR133A to HSV1 infected 293T cells (FIG. 5D-2). These studies further support that this conserved Arg site is necessary for PILRα binding to its ligands.

To better quantify the binding of WT versus Arg mutant PILRα binding to various ligands, we performed SPR analysis. The WT hPILRα-Fc and hPILRα R126-Fc were immobilized on a chip and their binding to hNPDC1, hCOLEC12, and mCD99 was compared (FIG. 5E). All three proteins showed strong binding with the WT hPILRα (FIG. 5E). However, little or no binding was observed with the mutant hPILRα, suggesting this conserved Arg site is necessary for binding of PILRα to its ligands (FIG. 5E).

Our data suggests that PILRα-ligand interactions require a conserved Arg motif on PILRα and specific sialylated decorations on the ligands. Since sialylation occur on many cell surface proteins, we predict PILRα ligands should be broadly expressed in various cells and hPILRαR126A/mPILRα133A should not bind to the natural ligands expressed on the surface of primary cells. To test this, we stained mouse and human hematopoietic cells with human or mouse PILRα-Fc. We found that both human and mouse PILRα-Fc bound to the majority of human PBMC (FIG. 5F-1). PILRα-Fc highly bound to human T cells and monocyte subsets (FIG. 5F-1 left, middle and lower panels). For murine cells, PILRα-Fc fusions highly bound to CD8⁺ peripheral T cells and thymocytes, as well as peripheral B cells (FIG. 5F-2,). However neither hPILRαR126A-Fc nor mPILRα133A-Fc can bind to human PBMCs (FIG. 5F-1), mouse thymocytes, or peripheral T and B cells (FIG. 5F-2). These results suggest that R126 in human PILRα and R133 in murine PILRα are required for their binding to cellular ligands expressed in primary cells.

Our study identifies the ARG126 (mouse ARG133) as a critical contact residue in hPILRα (FIG. 5A). Others have shown that TRP139 is also important in mediating hPILRα interaction with its ligand gB. Fan et al. (2010) J Virol 84, 8664-8672. Given the similarities of the binding residues between PILRα and Siglec family, we built a homology model of PILRα to gain insights into its contact residues.

The PILRα Sequence was aligned with the N terminal of the mouse Sialoadhesion from 1QFO.PDB using MOE2010.10 Protein Align application. Blosum62 was used as the alignment matrix, with tree-based build-up, Gap Start penalty of 7 and Gap Extend penalty of 1, Iteration limit of 100 and Failure limit of 10. The aligned structures were used to build a homology model using the Homology Model application in MOE2010.10. The crystal structure of the first chain in the SIGLEC1 was used as the template, while the ligand atoms were used as the ‘environment’ during model building. May et al. (1998) Mol Cell 1, 719-728; Munday and Crocker (1999) J Leukoc Biol 66, 705-711. The C, N-terminal outgaps were not built. A total of 25 models were built with fine minimization, and the final model was put through the Protonate3D procedure to detect the correct protonation states and was finely minimized at the end. The Merck force field (MMFF94x) with Born solvation method was used to reproduce the small molecule interactions of the active site. The active site-ligand interactions of SIGLEC1 and those of the PILRα model were rendered using the Ligand Interaction Diagram application of MOE2010.10. Clark and Labute (2007) J Chem Inf Model 47, 1933-1944. The active site-ligand contacts are coded according to Table 1.

TABLE 1 Ligand contacts of Siglec-1 and the PILRα model. Siglec-1 PILRα Contact Active Site Ligand Active Site Ligand Code Residue/Atom Residue/Atom Residue/Atom Residue/Atom Comments C01 TRP2.Ring SIA201.C11 TYR33.Ring SIA201.C11 3.6 Å vs 5.4 Å C02 TYR44.OH GAL202.O6 ARG74.NH1/NH2 GAL202.O6 [1] C03 ARG97.NH1 SIA201.O1A ARG126.NH1 SIA201.O1A identical C04 ARG97.NH2 SIA201.O1B ARG126.NH2 SIA201.O1B identical C05 SER103.O SIA201.O4 THR131 SIA201.O4 2.8 Å vs 4.8 Å C06 ARG132 SIA201.O4/O10 Unique to PILRα C07 ASN104.CA SIA201.O4 GLN137.OE1 SIA201.N5 5 Å vs 4.4 Å C08 ARG105.O SIA201.N5 GLN138.O SIA201.N5 Identical C09 ARG105.NE SIA201.O1A GLN138 SIA201.O1A Identical C10 TRP106.Ring SIA201.C9 TRP139.Ring SIA201.C9 Identical C11 LEU107.O SIA201.O9 GLN140.O SIA201.O9 Identical C12 LEU107.N SIA201.O8 GLN140.N SIA201.O8 Identical C13 LEU107.CD2 GAL202.O6 GLN140.NE2 GAL202.O6 3.8 Å vs 2.8 Å C14 ASP108 SER141 [2] Ligand atoms according to crystal structure (1QFO.pdb). Some corresponding atomic distance are shown under comments for Siglec-1 and PILRα respectively. [1] Contact possible via altered conformation; [2] Both residues about 5 Å away from SIA201.O9.

Despite the low sequence identity between SIGLEC1 and PILRα (FIG. 5A), it is clear that most of the contacts are strikingly similar, even when the active site residues are not identical. First, the C03, C04 contacts (SIGLEC1.ARG97 versus PILRα.ARG126) and the C10 contact (SIGLEC1.TRP106 versus PILRα.TRP139) are identical. SIGLEC1.SER103 and PILRα.THR131 both accept a hydrogen bond via their backbone carboxylate oxygens from the ligand Sialic acid 04 (contact C05). The backbone carboxylate oxygen of SIGLEC1.ARG105 and that of PILRα.GLN138 accept a hydrogen bond from N5 of the ligand sialic acid (contact C08). For contact C09, the guanidine nitrogen of this Arg in SIGLEC1 interacts with the carboxylate of ligand sialic acid O1A (4.1 Å), while the side chain amino in the corresponding residue (PILRα.GLN138) has the same contact, only slightly weaker (4.7 Å). The peptidic backbone of SIGLEC1.LEU107 and the corresponding PILRα.GLN140 have identical contacts with the ligand; their amino group contacts the ligand sialic acid O8 (coded as C11) and their carboxylate oxygen can accept a hydrogen bond from the ligand Sialic acid O9 (coded as C12). In comparison, the side chain amide group of PILRα.GLN140 is able to contact ligand galactose O6, which in SIGLEC1, the non-polar side chain of LEU108 has a non-favorable interaction with the ligand. This contact is coded as C13. The hydroxyl group of Tyrosine 44 of SIGLEC1 donates a hydrogen bond to O6 of the ligand galactose (contact code C02). The corresponding residue in PILRα (Arg73) cannot make this contact in the bent conformation observed in the model, where it seems to prefer to hydrogen bond with PILRα.PHE124.O but manual rotation of the side chain along CG-CD can restore not one but two hydrogen bonds with the same oxygen in the ligand (galactose O6) with slight repulsion from PILRα.GLN140.NE2. Overall, it is likely that this contact (C02) can be present in PILRα, even stronger than SIGLEC1. Similarly, while SIGLEC1.TRP2 has proton-Pi interaction with the ligand Sialic acid C11 with the distance of 3.6A, the base conformation of the corresponding residue (PILRα.TYR33) is slightly further (5.4A), but a 30 degree rotation of the tyrosine side chain along CA-CB makes perfect overlay of the two aromatic systems, suggesting that the two receptors might have nearly identical contacts at this site too (C01). The PILRα.ARG132 which has no corresponding residue in SIGLEC1 crystal structure and shows no interaction with the ligand in the base conformation. However, after rotating the side chain along Cα-Cβ or Cβ-Cγ, the side chain can make hydrogen bonds with the ligand sialic acid O4, O10. These interactions (C06) are unique to PILRα suggesting that distinctive features exist between SIGLEC1 and PILRα receptors. It is interesting to note that the conserved interaction residues are predominantly located in carboxyl end of PILRα between 123 to 142 amino acids (FIG. 5A, arrows). In summary, PILRα active site is very similar to the SIGLEC1, with at least two additional hydrogen bonding contacts (Arg132, Gln140). These residues are exposed to solvent, even in the complex, suggesting possible contacts with the protein domains of other ligands.

Despite only 41% amino acid identity between the human and mouse PILRα ECDs, we find that both proteins can still interact with similar ligands and primary cells types. This suggests that a conserved interacting domain has been selected during evolution. Correspondingly, the alignment of PILRα sequences points to high degree of conservation among potential contact residues (FIG. 5A) some of which are shared with Siglecs family of receptors) (Arginine 126, Tryptophane 139). Tabata et al. (2008) J Biol Chem 283, 8893-8901. Our studies demonstrate that one common binding mechanism involves the recognition of one or several sialic acid modifications in all ligands by the conserved arginine site in mouse and human PILRα. We have come to this conclusion by showing that ligand fusion proteins including mCD99-Fc, hNPDC1-Fc and hCOLEC12-his do not bind to hPILRαR126A and mPILRαR133A expressed on cell surface while they all bind to the wild type human or mouse PILRα (FIG. 5B). Second, we find that mutated fusion proteins hPILRαR126A-Fc and mPILRαR133A-Fc do not bind to cell surface expressed ligands including mCD99, hNPDC1, hCOLEC12 and HSV1 gB (FIG. 5C-D-2). Third, our surface plasmon resonance analysis clearly shows that only wild type hPILRα-Fc but not mutated hPILRαR126A-Fc can bind to various ligands. Finally, hPILRαR126A-Fc and mPILRαR133A-Fc fail to interact with natural ligand(s) expressed in primary hematopoietic cells (FIG. 5F), suggesting this conserved Arg site is required for PILRα binding to all nature ligands. PILRα's uniform binding interaction mode by sialic acid recognition might have evolved to trigger a conserved signaling pathway and functional outcome depending on which ligand(s) are binding to PILRα. The convergence of PILRα sequences across different species may be indicative of receptor genes that are responding to evolutionary pressure provided by pathogens or unknown ligands.

A highly conserved and essential arginine residue (Arg97 in SIGLEC1) contacts the carboxylate group of sialic acid and two tryptophans that interact with the N-acetyl and glycerol moieties of N-acetyl neuraminic acid. May et al. (1998) Mol Cell 1, 719-728; Munday et al. (1999) J Leukoc Biol 66, 705-711. Our functional data with arginine-mutated PILRα is consistent with a model in which Siglecs and PILRα have some similarity in their ligand interaction domains. Our analysis identifies arginine 126 as a key contact residue in hPILRα (Arg 133 in mPILRα) and its location appears to be critical to mediate PILRα interaction with sialic acid on gB as well as other ligands. Further support of this model is provided by recent data that the Tryptophan-139 residue is also critical for human PILRα binding to HSV1 gB. Fan et al. (2010) J Virol 84, 8664-8672. This Tryptophan-139 is not present in PILRβ and the converse mutation of L139W in PILRβ does not confer the ability to mediate binding to HSV1 gB, suggesting that there are other amino acids that play a role in the binding of PILRα with gB. Fan et al. (2010) J Virol 84, 8664-8672. Based on known structure of SIGLEC1:sialic acid complex, it is possible to speculate on how PILRα might recognize sialic acid containing ligands and rationalize the results of previously reported PILRα mutations. May et al. (1998) Mol Cell 1, 719-728. The homology model of the structure of PILRα identifies active site residues that are almost identical between the Siglecs and PILRα. In the absence of a crystal structure for PILRα, high similarities among the active site-ligand contacts between PILRα and SIGLEC1, even in case of non-identical residues allows us to confide in the homology model, and provides us with an expanded hypothesis for ligand binding of PILRα which can further analyzed via additional mutation studies. Structural resolution of PILRα in the context of sialylated proteins will be necessary to define the interaction domains. SIGLEC1 and multiple CD33-related Siglecs can interact with sialic acids on pathogens such as N. meningitidis, C. jejuni, group B Streptococcus and Trypanosoma cruzi. Crocker et al. (2007) Nat Rev Immunol 7, 255-266. It will be interesting to know whether PILRα could also directly interact with similar pathogens, which are known to carry sialylated sugar modifications in their surface structures.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. 

1. A method for modulating the cell surface interaction between a ligand and PILR-alpha comprising contacting cells with an effective amount of an agent that may modulate the interaction between said ligand and PILR-alpha, wherein the ligand is selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
 2. The method of claim 1, wherein the cells express PILR-alpha.
 3. The method of claim 1, wherein the cells express NPDC1.
 4. The method of claim 1, wherein the cells express COLEC12.
 5. The method of claim 1, wherein the cells express ETBR.
 6. The method of claim 1, wherein the cells express CLEC4G.
 7. The method of claim 1, wherein the cells express BR3.
 8. The method of claim 1, wherein the cells express MAG.
 9. The method of claim 1, wherein the cells express IL-2Ra.
 10. The method of claim 1, wherein the cells express FceRII.
 11. The method of claim 1, wherein the cells express LRRTM4.
 12. The method of claim 1, wherein the cells express DAG1.
 13. The method of claim 1, wherein the cells express APLP1.
 14. The method of claim 1, wherein the cells express PTPRN.
 15. The method of claim 1, wherein the cells express WDR31.
 16. The method of claim 1, wherein the cells express PSS8.
 17. The method of claim 1, wherein the cells express SIGLEC7.
 18. The method of claim 1, wherein the cells express IL15-RA.
 19. An agent for modulating the interaction between PILR-alpha and a ligand selected from NPDC1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
 20. A method for enhancing pathogen clearance in a subject comprising blocking the interaction between PILRα PILRα and a surface entry protein on the pathogen.
 21. (canceled)
 22. The method of claim 20, wherein said blocking comprises administering an antagonist of said PILRα to said subject, wherein said antagonist contacts PILRα at an epitope comprising one or more PILRα residues selected from the group consisting of Tyr33, Arg126, Thr131, Arg132, Gln138, Trp139, and Gln140.
 23. The method of claim 22, wherein said epitope comprises a PILRα residue Arg126.
 24. The method of claim 22, wherein said epitope comprises a PILRα residue Arg132.
 25. The method of claim 22, wherein said epitope comprises a PILRα residue Gln140.
 26. The method of any one of claims 23-25, wherein said epitope comprises the PILRα residues Arg126, Arg132, and Gln140.
 27. The method of claim 26, wherein said epitope consists of PILRα residues Tyr33, Arg126, Thr131, Arg132, Gln138, Trp139, and Gln140.
 28. The method of claim 20, wherein the pathogen is selected from listeria, herpes simplex virus-1, Neisseria meningitis, Haemophius influenzae, group B streptococcus, Campylobacter jejuni, Staphylococcus aureus, and Escherichia coli.
 29. The method of claim 22, wherein said antagonist is selected from the group consisting of antibodies and antigen-binding fragments thereof; proteins; peptides; glycoproteins; glycopeptides; glycolipids; polysaccharides; oligosaccharides; nucleic acids; bioorganic molecules; peptidomimetics; pharmacological agents and their metabolites; transcriptional and translation control sequences; small molecule inhibitors, fusions proteins, receptor molecules, and derivatives which bind specifically to a protein thereby sequestering its binding to its target; antisense molecules; RNA aptamers; and ribozymes.
 30. The method of claim 20, wherein said blocking comprises administering an antagonist of a surface entry protein comprising a sialidated glycan motif.
 31. The method of claim 20 or 30, wherein said blocking results in at least a 20% decrease in said interaction between said PILRα and said surface entry protein.
 32. The method of claim 31, wherein said blocking results in at least a 50% decrease in said interaction between said PILRα and said surface entry protein.
 33. The method of claim 32, wherein said blocking results in at least a 70% decrease in said interaction between said PILRα and said surface entry protein.
 34. A method of treating a subject having an inflammatory disease, said method comprising administering an effective amount of an antagonist of PILRα to said subject, wherein said antagonist contacts PILRα at an epitope comprising one or more PILRα residues selected from the group consisting of Tyr33, Arg126, Thr131, Arg132, Gln138, Trp139, and Gln140.
 35. The method of claim 34, wherein said epitope comprises PILRα residue Arg126.
 36. The method of claim 34, wherein said epitope comprises PILRα residue Arg132.
 37. The method of claim 34, wherein said epitope comprises PILRα residue Gln140.
 38. The method of any one of claims 35-37, wherein said epitope comprises PILRα residues Arg126, Arg132, and Gln140.
 39. The method of claim 38, wherein said epitope consists of PILRα residues Tyr33, Arg126, Thr131, Arg132, Gln138, Trp139, and Gln140.
 40. The method of claim 34, wherein said antagonist is selected from the group consisting of antibodies and antigen-binding fragments thereof; proteins; peptides; glycoproteins; glycopeptides; glycolipids; polysaccharides; oligosaccharides; nucleic acids; bioorganic molecules; peptidomimetics; pharmacological agents and their metabolites; transcriptional and translation control sequences; small molecule inhibitors, fusions proteins, receptor molecules, and derivatives which bind specifically to a protein thereby sequestering its binding to its target; antisense molecules; RNA aptamers; and ribozymes.
 41. The method of claim 34, wherein said administering results in an inhibition of the activity of said PILRα by at least 20%.
 42. The method of claim 41, wherein said administering results in an inhibition of the activity of said PILRα by at least 50%.
 43. The method of claim 42, wherein said administering results in an inhibition of the activity of said PILRα by at least 70%. 